Lei et al. Cell Death and Disease (2020) 11:331 https://doi.org/10.1038/s41419-020-2513-1 Cell Death & Disease ARTICLE Open Access SOX1 promotes differentiation of nasopharyngeal carcinoma cells by activating retinoid metabolic pathway Xin-Xing Lei1,YunLiu2, Jin-Xing Wang3,QianCai2,MinYan1,Hui-PingHe1,QuentinLiu1,3,Zi-JieLong3 and Zhong Guan2 Abstract Undifferentiation is a key feature of nasopharyngeal carcinoma (NPC), which presents as a unique opportunity for intervention by differentiation therapy. In this study, we found that SOX1 inhibited proliferation, promoted differentiation, and induced senescence of NPC cells, which depended on its transcriptional function. RNA-Seq- profiling analysis showed that multiple undifferentiated markers of keratin family, including KRT5, KRT13, and KRT19, were reduced in SOX1 overexpressed NPC cells. Interestingly, gene ontology (GO) analysis revealed genes in SOX1 overexpressed cells were enriched in extracellular functions. The data of LC/MS untargeted metabolomics showed that the content of retinoids in SOX1 overexpressed cells and culture medium was both higher than that in the control group. Subsequently, we screened mRNA level of genes in retinoic acid (RA) signaling or metabolic pathway and found that the expression of UDP-glucuronosyltransferases was significantly decreased. Furtherly, UGT2B7 could rescue the differentiation induced by SOX1 overexpression. Inhibition of UGTs by demethylzeylasteral (T-96) could mimic SOX1 to promote the differentiation of NPC cells. Thus, we described a mechanism by which SOX1 regulated 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; the differentiation of NPC cells by activating retinoid metabolic pathway, providing a potential target for differentiation therapy of NPC. Introduction offer new opportunities for treating NPC. Recently, Nasopharyngeal carcinoma (NPC) is endemic in remarkable achievements have been made by differ- South-Eastern Asia, and northern Africa. At present, the entiation therapy in the treatment of some types of main treatment approaches for NPC include radio- tumors3,4. 95% of NPC is pathologically undifferentiated therapy, chemotherapy, and targeted therapy. Radiation squamous cell carcinoma, which make NPC an appro- therapy is the firstchoiceforthetreatmentofNPC. priate model for differentiation therapy. However, it is inevitable that nasopharyngeal mucosa SOX1 encodes a member of the SOX (SRY-related and tissue will be damaged after radiotherapy1,2. Thus, high mobility group box) family of transcription factors more effective and less toxic regimens are needed to involved in the regulation of embryonic development – and determination of the cell fate5 7.SOX1shares similar biochemical properties and functions with SOX2 Correspondence: Quentin Liu ([email protected])or or other members, but the different affinity to target Zi-Jie Long ([email protected]) or Zhong Guan ([email protected]) genes, post-transcriptional modifications and interac- 1Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, 510060 Guangzhou, China tions with other co-factors lead to different functions in – 2Department of Otorhinolaryngology, Sun Yat-sen Memorial Hospital, Sun Yat- distinct biological contexts8 10. SOX proteins bind sen University, 510120 Guangzhou, China sequence-specifically to DNA by a high-mobility group Full list of author information is available at the end of the article These authors contributed equally: Xin-Xing Lei, Yun Liu (HMG) domain that allows them to function as Edited by A. Stephanou © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Lei et al. Cell Death and Disease (2020) 11:331 Page 2 of 15 transcription factors8,11. Gene silencing in cancer is V5-tagged SOX1, ΔHMG SOX1, or Δ246-391 SOX1. The associated with promoter hypermethylation12 and SOX1 primers used for gene cloning and plasmid construction hypermethylation was detected in primary tumor tis- are listed in Supplementary Table S1. sues13. SOX1 inhibits cell proliferation, reduces migra- tion and invasion ability, and induces differentiation of Lentiviral production, infections, and cell line generation – tumor cells14 17. Recently, we found that SOX1 was Lentivirus was produced by transient transfection by associated with decreased expression of undifferentiation Lipofectamine 2000 (Invitrogen) in human embryonic markers (KRT13 and KRT19) in NPC cells16. However, kidney (HEK) 293T cells using with a second-generation the mechanism by which SOX1 promotes NPC cell dif- lentiviral vector system. All virus-containing medium was ferentiation has not been established. mixed with 8 μg/mL polybrene (Sigma-Aldrich). Virus Retinoic acid (RA), especially all-trans-retinoic acid produced from pLVX-Tet3G plasmid was used to infect (ATRA), is the most potent natural metabolite of vitamin wild type HONE1 or CNE2 cells to construct HONE1- A. RA is involved in a variety of biological functions Tet-On or CNE2-Tet-On stable cell lines. Cells were including embryogenesis, cell differentiation, and apop- selected in 1 mg/mL G418 for at least 2 weeks. Subse- tosis18,19. Retinoids induce differentiation and/or apop- quently, HONE1-Tet-On and CNE2-Tet-On cell lines tosis in tumor cells and show anti-proliferative and anti- were infected by virus produced from pLVX-TRE-(X) oxidant activity, which have great potential as therapeutic plasmids and selected by 2 μg/mL puromycin for 6 days. or preventive agents. The distribution and level of RA in HONE1TRE-SOX1 and CNE2TRE-SOX1 cells infected by virus embryonic tissues are tightly controlled by oxidation produced from pLVX-UGT1A6 or pLVX-UGT2B7 plas- through retinol and retinal dehydrogenases and by mids were selected by 12 μg/mL blasticdin for 7 days. hydroxylation via specific cytochrome P450 family 26 enzymes (CYP26s)20,21. Cell culture and doxycycline induction UDP-glucuronosyltransferases (UGTs) are phase II The HONE1 and CNE2 cell lines were obtained from metabolism isoenzymes that are found to be active in Dr.Chao-NanQian(SunYat-senUniversity,Guangz- liver, kidneys, epithelial cells of the lower gastrointestinal hou, China). Wild type cell lines and their lentiviral- tract and brain20,22. Although the characterizations of infected stable cell lines were all maintained in RPMI UGT gene family members were identified, their roles in 1640 (Invitrogen) supplemented with 10% fetal bovine clearance and homeostasis of endogenous substrates are serum (FBS, Hyclone). The cells were incubated at 37 °C still insufficiently understood. Previous study proves that in a humidified chamber containing 5% CO2.HONE1- retinoids and their analogs are glucuronidated by Tet-On-(X) and CNE2-Tet-On-(X) cell lines were trea- UGTs20,23,24. Excess retinoids can also inhibit the ted with 2 μg/mL doxycycline (Medchem Express) to expression of UGTs, indicating the existence of regulatory induce overexpression of SOX1 or its mutant/ loops and complexity25. truncated form. In the present study, we showed that overexpression of SOX1 promoted NPC cells to differentiate via its tran- Cell viability assay scriptional function. These new data also established a Cells were seeded onto 96-well plates at an initial mechanism that retinoids were accumulated by SOX1, density of 2 × 103 cells/well. At specified time points, contributing to the differentiation of NPC cells, which 10 μL of CCK-8 solution was added to each well of the offered a novel approach for NPC differentiation therapy. plate. Then the plate was incubated for another 2 h. Cell viability was determined by scanning with a microplate Materials and methods reader at 450 nm. Plasmid constructs The plasmids encoding human SOX1 (wild type, WT), Immunofluorescence staining mutant SOX1 (SOX1mut_2), V5-tagged SOX1, ΔHMG Cells were fixed in 4% paraformaldehyde at room SOX1, and Δ246-391 SOX1 were generated by PCR temperature for 10 min and permeabilized in 0.5% Tri- amplification and subcloned into the pLVX-TRE3G ton X-100 in PBS for 10 min. Slides were incubated with expression vector; UGT1A6 and UGT2B7 were sub- the primary antibody (1:200 dilution) overnight. Immune cloned into pLVX expression vector; promoters of complexes were stained with the secondary antibody UGT1A6 and UGT2B7 were subcloned into pGL3-basic conjugated to Alexa-488 or Alexa-546 (Invitrogen, 1:200 vector using the ClonExpress II One Step Cloning Kit or dilution) at room temperature for 1 h. Nuclei were ClonExpress MultiS One Step Cloning Kit (Vazyme) stained with DAPI (Sigma-Aldrich) and viewed with an according to the manufacturer’s instructions. The new Olympus IX71 microscope. The following antibodies plasmid was named